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Formalin, an aqueous solution of formaldehyde, has been the gold standard for fixation of histological samples for over a century. Despite its considerable advantages, growing evidence points to objective toxicity, particularly highlighting its carcinogenicity and mutagenic effects. In 2016, the European Union proposed a ban, but a temporary permission was granted in consideration of its fundamental role in the medical-diagnostic field. In the present study, we tested an innovative fixative, glyoxal acid-free (GAF) (a glyoxal solution deprived of acids), which allows optimal tissue fixation at structural and molecular level combined with the absence of toxicity and carcinogenic activity. An open-label, non-inferiority, multicentric trial was performed comparing fixation of histological specimens with GAF fixative vs standard phosphate-buffered formalin (PBF), evaluating the morphological preservation and the diagnostic value with four binary score questions answered by both the central pathology reviewer and local center reviewers. The mean of total score in the GAF vs PBF fixative groups was 3.7 ± 0.5 vs 3.9 ± 0.3 for the central reviewer and 3.8 ± 0.5 vs 4.0 ± 0.1 for the local pathologist reviewers, respectively. In terms of median value, similar results were observed between the two fixative groups, with a median value of 4.0. Data collected indicate the non-inferiority of GAF as compared to PBF for all organs tested. The present clinical performance study, performed following the international standard for performance evaluation of in vitro diagnostic medical devices, highlights the capability of GAF to ensure both structural preservation and diagnostic value of the preparations.

Formaldehyde-based fixatives, particularly neutral-buffered formalin (NBF), are widely used in histology owing to their strong protein cross-linking capacity and reliable preservation of cytoarchitecture. However, formaldehyde is toxic and carcinogenic, motivating the search for safer alternatives. Here, we systematically evaluated lactic acid (LA) as a potential formaldehyde-free fixative for murine brain tissues. Brains were fixed either by immersion or by transcardial perfusion followed by immersion using LA at varying concentrations, pH-adjusted LA, or phosphate-buffered saline (PBS), and were compared with NBF. Histomorphological preservation was assessed using qualitative evaluation, semi-quantitative scoring, and quantitative crack-area measurements. Across all conditions, NBF provided the best overall tissue preservation. LA fixation showed clear concentration-dependent effects: low concentrations resulted in poor structural integrity, whereas higher concentrations (LA10–LA20) achieved moderate preservation of cytoarchitecture, reduced cracking, and acceptable cellular morphology. White-matter integrity remained inferior to NBF, consistent with the lipid-rich composition of myelin and the predominantly protein-denaturing, non-cross-linking fixation properties of LA. pH-adjustment experiments indicated that fixation efficacy partially depended on acidity. Notably, LA20 delivered by transcardial perfusion substantially improved tissue preservation compared with immersion alone and outperformed PBS. Although LA cannot fully replace NBF, high-concentration LA might represent an alternative for selected neurohistological applications.

This technical report describes a method to clear fixed brain tissues while allowing for fluorescent dye tracing and retaining cellular morphology. The authors demonstrate the utility of the technique by obtaining a wiring diagram for sister mitral cells. We report a water-based optical clearing agent, SeeDB, which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. Our method maintained a constant sample volume during the clearing procedure, an important factor for keeping cellular morphology intact, and facilitated the quantitative reconstruction of neuronal circuits. Combined with two-photon microscopy and an optimized objective lens, we were able to image the mouse brain from the dorsal to the ventral side. We used SeeDB to describe the near-complete wiring diagram of sister mitral cells associated with a common glomerulus in the mouse olfactory bulb. We found the diversity of dendrite wiring patterns among sister mitral cells, and our results provide an anatomical basis for non-redundant odor coding by these neurons. Our simple and efficient method is useful for imaging intact morphological architecture at large scales in both the adult and developing brains.

RNA and DNA analyses from paraffin-embedded tissues (PET) are an important diagnostic tool for characterization of a disease, exploring biomarkers and treatment options. Since nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissue are of limited use for molecular analyses due to chemical modifications of biomolecules alternate, formalin-free fixation reagents such as the PAXgene Tissue system are of evolving interest. Furthermore, biomedical research and biomarker development critically relies on using long-term stored PET from medical archives or biobanks to correlate molecular features with long-term disease outcomes. We therefore performed a comparative study to evaluate the effect of long term storage of FFPE and PAXgene Tissue-fixed and paraffin-embedded (PFPE) tissue at different temperatures on nucleic acid stability and usability in PCR. Matched FFPE and PFPE human tissues from routine clinical setting or rat tissues from a highly controlled animal model were stored at room temperature and 4°C, as well as in case of animal tissues frozen at -20°C and -80°C. RNA and DNA were extracted in intervals for up to nine years, and examined for integrity, and usability in quantitative RT-PCR (RT-qPCR) or PCR (qPCR) assays. PET storage at room temperature led to a degradation of nucleic acids which was slowed down by storage at 4°C and prevented by storage at -20°C or -80°C. Degradation was associated with an amplicon length depending decrease of RT-qPCR and qPCR efficiency. Storage at 4°C improved amplifiability in RT-qPCR and qPCR profoundly. Chemically unmodified nucleic acids from PFPE tissue performed superior compared to FFPE tissue, regardless of storage time and temperature in both human and rat tissues. In conclusion molecular analyses from PET can be greatly improved by using a non-crosslinking fixative and storage at lower temperatures such as 4°C, which should be considered in prospective clinical studies.

Purpose Numerous fixative solutions are available but many are not amenable to the histomorphological preservation of retinae. The investigators specifically focused on retinal histological studies, which rather than 4% formaldehyde (FA), often use Davidson’s fixative. However the latter has its limitations. The purpose of this study was to produce a new fixative which maintains retinae closer to the in vivo conditions. Study design Experimental design. Methods Four fixative formulations (4% paraformaldehyde, Davidson’s fixative, modified Davidson’s fixative and an in-house fixative – TB-Fix) were tested on retinae and the outcomes on histomorphology and immunohistochemical staining for selected antigenic markers was compared. Results TB-Fix markedly improved morphological detail following hematoxylin and eosin staining, most importantly eliminating the spongiform appearance in the plexiform layer and the swelling of somata (including Müller cells), when compared to FA, Davidson’s fixative and its modified version. Retinal samples fixed with TB-Fix or FA showed comparable results in immunohistological staining for neurons and glia in the retina. Importantly, while the whole eye fixed with FA collapsed in shape and induced artificial retinal detachment, the eye fixed with TB-Fix avoided deformation and detachment. Furthermore, we found that TB-Fix also prevented detachment from the culture plate when used to fix HEK293 cells, which are known to detach from the plate easily. Conclusion It was demonstrated that TB-Fix provides an overall improvement in the preservation of retinal morphology and chemical composition.

Demonstration of glycogen can be done in different lesions and is considered diagnostically significant, mainly in some tumors. Glycogen staining is affected by the type of fixative, the temperature of fixation, and the staining technique.Grocott’s methenamine (hexamine) silver technique quality was assessed after four different types of fixatives at two different temperatures [Bouin’s solution, 10% neutral buffered formalin (NBF), 80% alcohol, and Rossman’s solution at room temperature (RT) and 4 °C, for 24 h]. These variables were studied to optimize this technique for glycogen demonstration. Archived paraffin blocks were used in this study. They were prepared from one rabbit’s liver, and 32 paraffin sections were prepared and stained with Grocott’s methenamine (hexamine) silver technique. Eighty percent alcohol provided the highest staining quality scores at both RT and 4 °C in comparison with the other fixatives. We concluded that 80% alcohol at 4 °C seems to be the fixative of choice for glycogen with the Grocott’s methenamine (hexamine) silver technique at the level of this study.

Kicking a person laying on the floor in the head is a crime whose forensic investigation could profit from additional microtraces capable of linking a suspected footwear, and by extension its owner, to the victim and their injuries. The transfer of hair fixatives (hair gel, hair wax, hair spray, hair foam, etc.) represents such a trace and was consequently practically evaluated throughout this study. This study consists of two parts: The first part, the differentiation study, encompasses the visual, and instrumental analysis of a variety of different hair fixatives to determine their analysability and differentiation potential. The visual examination was conducted using alternate light sources and filter lenses. Subsequently, the instrumental analysis was carried out, whereby the focus lay on Fourier Transform Infra-red (FT-IR) spectroscopy and Raman spectroscopy. The second part is comprised of different experiments including a test-transfer and pendulum experiments to assess the process and the potential variables of the transfer of hair fixative traces between hair and fabric shoes during a kick. This helped to determine the effect of the kick strength and the behaviour of differing hair products. Retrieval methods to secure hair fixative traces of footwear and from the hair of a victim were developed. These were subsequently tested out on an acute case example.. ●Analysing the process of kicking a person in the head.●Locating, identifying, and extracting hair fixatives from shoes of a suspected offender, and from the hair of a victim.●Comparing hair fixative traces from shoes with those of the hair of the victim.●Distinguishing hair fixatives from different branches visually and instrumentally.

Proper fixing and long-term preservation of entomological evidence are essential in collections and research and crucial in applied fields such as forensic entomology. Incorrectly stored samples may lose important morphological features over time, rendering molecular analyses exceedingly difficult. The most effective method for preserving soft samples such as larvae is fluid preservation. It uses a combination of a wide range of fixatives and storage fluids. However, very little comparative work has been done to determine the effects of long-term storage on sample quality in terms of color, shape, and DNA stability. Moreover, the current golden standard in forensic entomology has been tailored for age estimation of larvae of Diptera, which differ from larvae of Coleoptera in morphology and subsequently in applied methods. We compared the effects of combinations of 6 commonly used fixatives and 6 commonly used storage fluids on midsized larvae of the forensically important beetle, Necrodes littoralis (Linnaeus, 1758), in terms of color, shape, and suitability for DNA analyses over a 2-yr period. We were looking for combinations that can preserve specimens in a satisfactory state, can be used on a regular basis, do not require advanced protection or skills of the personnel, and are not toxic or too harmful to the environment. We found not only several methods that scored significantly better in the tested parameters compared with the golden standard but also several common methods that should be avoided. The effects of agents on each tested category are discussed in detail.

Archived tissues from previously completed prospective trials represent invaluable resource for biomarker development. However, such specimens are often stored as sections on glass slides, in which RNA is severely degraded due to prolonged air exposure. We evaluated whether a proportion of archived sectioned formalin-fixed paraffin-embedded (AS-FFPE) tissues yield transcriptome profiles comparable to freshly cut (FC) FFPE tissues, which can be used for retrospective class prediction analysis. Genome-wide transcriptome profiles of 6 to 7-year-old AS-FFPE tissue sections (generated from 5 to 16-year-old blocks) of 83 hepatocellular carcinoma (HCC) and 47 liver cirrhosis samples were generated by using whole-genome DASL assay (Illumina) and digital transcript counting (nCounter) assay (NanoString), and gene signature-based prediction of HCC subclasses and prognosis was compared with previously generated FC-FFPE profiles from the same tissue blocks. RNA quality and assay reproducibility of AS-FFPE RNA were comparable to intermediate to poor quality FC-FFPE samples (RNA Integrity Number: up to 2.50, R-square for technical replicates: up to 0.93). Analyzable transcriptome profiles were obtained in 64 (77%) HCC and 36 (77%) cirrhosis samples. Statistically more confident predictions based on random resampling-based method (nearest template prediction) were obtained in 37 (58%) HCC and 13 (36%) cirrhosis samples. Predictions made in FC-FFPE profiles were reproduced in 36 (97%) HCC and 11 (85%) cirrhosis AS-FFPE profiles. nCounter assay was tested in 24 cirrhosis samples, which yielded confident prediction in 15 samples (63%), of which 10 samples (67%) showed concordant predictions with FC-FFPE profiles. AS-FFPE tissues yielded poorer quality RNA and transcriptome profiles compared to FC-FFPE tissues. Statistically more confident class prediction was feasible in 37 of 83 HCC samples and 13 of 47 cirrhosis samples. These results suggest that AS-FFPE tissues can be regarded as a resource for retrospective transcriptome-based class prediction analysis when they are the only available materials.

The application of mass spectrometry imaging (MSI) for the study of spatiotemporal alterations of the metabolites in tumors has brought a number of significant biological results. At present, metabolite profiling based on MSI is typically performed on frozen tissue sections; however, the majority of clinical specimens need to be fixed in tissue fixative to avoid autolysis and to preserve antigenicity. In this study, we present the global impacts of different fixatives on the MS imaging of gastric cancer tissue metabolites. The MSI performances of 17 kinds of metabolites, such as amino acids, polyamines, cholines, organic acids, polypeptides, nucleotides, nucleosides, nitrogen bases, cholesterols, fatty acids, and phospholipids, in untreated, 10% formalin–, 4% paraformaldehyde–, acetone-, and 95% ethanol–fixed gastric cancer tissues were thoroughly explored for the first time. Furthermore, we also investigated the spatial expressions of 6 metabolic enzymes, namely, GLS, FASN, CHKA, PLD2, cPLA2, and EGFR, closely related to tumor-associated metabolites. Immunohistochemical staining carried out on the same tissue sections‚ which have undergone MSI analysis‚ suggests that enzymatic characterization is feasible after metabolite imaging. Combining the spatial signatures of metabolites and pathway-related metabolic enzymes in heterogeneous tumor tissues offers an insight to understand the complex tumor metabolism.